Novel system for the sequential, directional cloning of multiple DNA sequences

ABSTRACT

A method which combines the use of polymerase chain reaction (PCR) or oligonucleotide linkers and restriction enzymes which cleave degenerate or variable recognition site sequences to allow the cloning of multiple DNA sequences into a vector is disclosed. In this invention, a plurality of unrelated DNA sequences may be directionally cloned within a single vector by adding onto the ends of the sequences, restriction sites with specific sequences which are cleaved by corresponding restriction endonucleases which recognize degenerate or variable recognition sites and which generate cohesive ends upon cleavage. The compatibility (or ability to anneal) of the cohesive ends on different DNA sequences is controlled by the choice of the nucleotide sequence within the recognition sequences of the restriction endonucleases, allowing the DNA sequences to be inserted or joined in any desired orientation. These restriction sites may be selectively incorporated onto either or both end of any DNA sequence of interest using oligonucleotidelinkers or using PCR by adding the restriction sites onto the termini of the 5′ and/or 3′ primers.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The invention relates to a novel system and method for thesequential, directional cloning of multiple DNA sequences into a singlevector.

[0003] 2. Description of the Prior Art

[0004] The directional ligation of multiple DNA sequences within vectorsis often hindered by the inability to force the orientation ofsubsequently ligated DNA fragments. This necessitates determination offragment orientation following each ligation event to select recombinantplasmids with the inserts in the correct orientation (Potter, 1996,Biotechniques, 21:198-200). In addition, when attempting to clone anumber of unrelated DNA fragments into a single host, the number ofusable restriction sites declines rapidly, due to the presence of thesites in the insert DNA(s). Although it is sometimes possible to insertmultiple genes into a single vector using a combination of availablemulti-cloning site (MCS) restriction sites (Jach et al., 1995, PlantJournal, 8:97-109; and Yamano et al., 1994, Biosci. Biotechnol.Biochem., 58:1112-1114), the process is often impractical. Moreover, theprocess is even more unreliable when attempting to directionally clonemore than two genes into the vector.

SUMMARY OF THE INVENTION

[0005] We have now discovered a method which combines the use ofpolymerase chain reaction (PCR) or oligonucleotide linkers andrestriction enzymes which cleave recognition site sequences that haveinternal degeneracy to allow the sequential, directional cloning ofmultiple DNA sequences into a DNA vector. In this invention, a pluralityof unrelated DNA sequences may be directionally cloned within a singlevector by adding onto the ends of the sequences, restriction-sites withspecific sequences which are cleaved by corresponding restrictionendonucleases which recognize degenerate or variable recognition sitesand which generate cohesive ends upon cleavage. The compatibility (orability to anneal) of the cohesive ends on different DNA sequences iscontrolled by the choice of the nucleotide sequence within therecognition sequences of the restriction endonucleases, allowing the DNAsequences to be inserted or joined in any desired orientation.

[0006] Generally, a recipient DNA (such as a vector) is provided whichhas a first restriction site having a degenerate recognition sequencewith a predetermined nucleotide sequence, and which upon digestion withits restriction enzyme generates cohesive ends. A DNA sequence to beinserted into the recipient DNA is provided with a different restrictionsite at each end, which also include degenerate recognition sequences.However, the nucleotide sequences of these degenerate recognitionsequences are selected such that upon digestion, they give rise to firstand second cohesive ends which are each complementary to only one of thecohesive ends on the recipient DNA. The first cohesive end on the insertDNA is only complementary to one cohesive end on the recipient DNA,while the second cohesive end on the insert DNA is only complementary tothe other cohesive end on the recipient DNA. Thus, the directionality ordesired orientation of the ligation of the inserted DNA to the recipientDNA or vector is ensured. Furthermore, by choosing such restrictionsites which are the same (cleaved by the same restriction enzyme), ordifferent (cleaved by different enzymes), the user may selectivelypredetermine if the functional restriction site is or is not regeneratedafter ligation. When the cohesive ends generated from two of the samerestriction sites are annealed, the functional restriction site will beregenerated. In contrast, the cohesive ends generated from two differentrestriction sites, although complementary, will not regenerate afunctional restriction site when annealed.

[0007] These restriction sites may be selectively incorporated onto theend(s) of any DNA sequence of interest using PCR by adding therestriction sites onto the termini of the 5′ and/or 3′ primers, or byadding linkers to the DNA sequence.

[0008] In this process, the first DNA sequence of interest may beinserted into-the vector using either the process of this invention, ora variety of known techniques, including ligation into the vector atrestriction sites generating blunt ends or cohesive ends, or acombination thereof. For instance, at least one end of the DNA sequencemay be provided with a restriction site generating a cohesive end uponcleavage, which may then be inserted into the vector at any site whichgenerates complementary cohesive ends.

[0009] To facilitate the ligation of additional DNA sequences, the firstDNA sequence (further) includes one of the above-mentioned restrictionsites having a degenerate recognition sequence adjacent (near) aselected end which also generates a cohesive end upon digestion with itscorresponding restriction enzyme. This should be different from anyother restriction sites present on the first DNA sequence, and should beunaffected by any initial restriction enzymes which may be used toinsert the first sequence into the vector. This site should also beinternal to any other different restriction sites used to insert thefirst DNA sequence into the vector to ensure that it is preserved.

[0010] The second DNA sequence of interest which is to be ligatedadjacent to (upstream or downstream) from the first sequence is alsoprovided with a restriction site adjacent to a selected first end thatis different from the restriction site on the first sequence, and has adegenerate recognition sequence which, upon cleavage with itscorresponding restriction enzyme, generates a cohesive end. Thenucleotide sequences of the degenerate regions in these restrictionsites (adjacent to the selected end of the first DNA sequence and thefirst end of the second DNA sequence) are selected such that thecohesive ends generated upon cleavage will be complementary to eachother. If further DNA sequences are to be inserted into the vectoradjacent to the second DNA sequence, the-second DNA sequence should alsoinclude an additional restriction site adjacent to its opposite orsecond end which is essentially the same as the above-mentionedrestriction site on the first end of the first DNA sequence. Moreover,because the restriction sites at the ends of the second DNA sequencegenerate asymmetric cohesive ends when cleaved, the directionality ororientation of the insertion into the vector may be readily controlledby selection of the restriction sites and the nucleotide sequences oftheir degenerate internal recognition regions.

[0011] Upon digestion of the restriction sites on the second DNAsequence and the restriction site on the selected end of the first DNAsequence (now contained within the recombinant plasmid) with theirrestriction enzymes, each of the ends on the cut vector will becompatible to only one of the ends on the second DNA sequence, ensuringdirectionality. Specifically, the digestion of the restriction site onthe first DNA sequence will generate overhangs on each end of the cutvector (one adjacent to the first DNA sequence and the other at theopposite end of the vector). The restriction site on the first end ofthe second DNA sequence will generate a cohesive end that is onlycomplementary to the cohesive end adjacent to the first DNA sequence(i.e., at the selected end of the first DNA sequence), while therestriction site on the second end of the second DNA sequence willgenerate a cohesive end which is only complementary to the cohesive endon the opposite end of the cut vector. Upon ligation of these overhangs,not only will the second DNA sequence be inserted into the vectoradjacent to the first DNA sequence in the desired orientation, but therestriction site at the second end of the second sequence will also beregenerated. Recreation of this restriction site will allow insertion ofa further DNA sequence.

[0012] Any number of additional DNA sequences of interest may then beinserted into the vector sequentially from the second DNA sequencefollowing the same protocol described for the second sequence.

[0013] In accordance with this discovery, it is an object of thisinvention to provide a method for directionally inserting multiple DNAsequences into a single DNA vector in a desired orientation.

[0014] It is also an object of this invention to provide a method forpreparing expression vectors containing a plurality of genes in adesired orientation for insertion into host cells and expression of allof the gene products therefrom.

[0015] Another object of this invention is to provide a method forcreating multi-gene cassettes which can be used as single intact unitsand transferred into other vectors or host cells.

[0016] Yet another object of this invention is to provide a method fortransforming-host cells with multiple genes using a singletransformation.

[0017] Other objects and advantages of this invention will becomereadily apparent from the ensuing description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1. Description of DraIII and SfiI linkers used todirectionally clone PCR products. Panel A depicts the sequence of theDraIII and SfiI linkers added to the termini of PCR primers. Panel Bshows the ligation of a DraIII/SfiI digested insert into a SfiI digestedvector.

[0019]FIG. 2. Diagram of pFSPME, an intermediate vector utilized inconstruction of chimeric crt genes. The Erwinia crt open reading frames(crtE, crtB, and citI) were subcloned into this vector (utilizing thePmeI and XhoI sites) in order to fuse the coding regions with Tri5promoter and terminator elements. The chimeric genes could then beexcised from the vector using Draone and Sfione primers. Details ofvector construction are described in the Experimental Protocol.

[0020]FIG. 3. Gene cassette construction strategy. pCRTEX1 was createdby cloning EcoRI digested insert#1 into EcoRI/SmaI digested BluescriptIIKS⁻. pCRTEX2 was created by directionally cloning DraIII/SfiI digestedinsert#2 into SfiI digested pCRTEX1; pCRTEX3 was subsequently created bycloning insert#3 into pCRTEX2. The gene cassette can be liberated fromthe vector sequences by NotI digestion, and cloned into alternatetransformation vectors.

[0021]FIG. 4. Transformation plasmids for Fusarium sporotrichioides.pA1L-E (top) and p4AL-I (center) contain the Aspergillus nidulans amdSgene under the control of the native A. nidulans amdS promoter (pA1L-E),or under the control of the Heterostrophus cochlibolus Promoter 1element (pA4L-I). pHL-J had the hygB gene driven by the Promoter 1element. The direction of crt gene transcription is denoted by the arrowdirection.

DEFINITIONS

[0022] The following terms are employed herein:

[0023] Cloning. The selection and propagation of (a) genetic materialfrom a single individual, (b) a vector containing one gene or genefragment, or (c) a single organism containing one such gene or genefragment.

[0024] Cloning Vector. A plasmid, virus, retrovirus, bacteriophage,cosmid, artificial chromosome (bacterial or yeast), or nucleic acidsequence which is able to replicate in a host cell, characterized by oneor a small number of restriction endonuclease recognition sites at whichthe sequence may be cut in a predetermined fashion, and which maycontain an optional marker suitable for use in the identification oftransformed cells, e.g., tetracycline resistance or ampicillinresistance. A cloning vector may or may not possess the featuresnecessary for it to operate as an expression vector.

[0025] Codon. A DNA sequence of three nucleotides (a triplet) whichcodes (through mRNA) for an amino acid, a translational start signal, ora translational termination signal. For example, the nucleotide tripletsTTA, TTG, CTT, CTC, CTA, and CTG encode for the amino acid leucine,while TAG, TAA, and TGA are translational stop signals, and ATG is atranslational start signal.

[0026] DNA Coding Sequence. A DNA sequence which is transcribed andtranslated into a polypeptide in vivo when placed under the control ofappropriate regulatory sequences. The boundaries of the coding sequenceare determined by a start codon at the 5′ (amino) terminus and atranslation stop codon at the 3′ (carboxy) terminus. A coding sequencecan include, but is not limited to, procaryotic sequences and cDNA fromeukaryotic mRNA. A polyadenylation signal and transcription terminationsequence will usually be located 3′ to the coding sequence.

[0027] DNA Sequence. A linear series of nucleotides connected one to theother by phosphodiester bonds between the 3′ and 5′ carbons of adjacentpentoses.

[0028] Expression. The process undergone by a structural gene to producea polypeptide. Expression requires both transcription of DNA andtranslation of RNA.

[0029] Expression Cassette. A nucleic acid sequence within a vectorwhich is to be transcribed, and a promoter to direct the transcription.The expression cassette may contain one or more unrelated DNA sequencesencoding one or more peptides of interest.

[0030] Expression Vector. A replicon such as a plasmid, virus,retrovirus, bacteriophage, cosmid, artificial chromosome (bacterial oryeast), or nucleic acid sequence which is able to replicate in a hostcell, characterized by a restriction endonuclease recognition site atwhich the sequence may be cut in a predetermined fashion for theinsertion of a heterologous DNA sequence. An expression vector has apromoter positioned upstream of the site at which the sequence is cutfor the insertion of the heterologous DNA sequence, the recognition sitebeing selected so that the promoter will be operatively associated withthe heterologous DNA sequence. A heterologous DNA sequence is“operatively associated” with the promoter in a cell when RNA polymerasewhich binds the promoter sequence transcribes the coding sequence intomRNA which is then in turn translated into the protein encoded by thecoding sequence.

[0031] Fusion Protein. A protein produced when two heterologous genes orfragments thereof coding for two different proteins not found fusedtogether in nature are fused together in an expression vector. For thefusion protein to correspond to the separate proteins, the separate DNAsequences must be fused together in correct translational reading frame.

[0032] Gene. A segment of DNA which encodes a specific protein orpolypeptide, or RNA.

[0033] Genome. The entire DNA of an organism. It includes, among otherthings, the structural genes encoding for the polypeptides of thesubstance, as well as operator, promoter and ribosome binding andinteraction sequences.

[0034] Heterologous DNA. A DNA sequence inserted within or connected toanother DNA sequence which codes for polypeptides not coded for innature by the DNA sequence to which it is joined. Allelic variations ornaturally occurring mutational events do not give rise to a heterologousDNA sequence as defined herein.

[0035] Hybridization. The pairing together or annealing of singlestranded regions of nucleic acids to form double-stranded molecules.

[0036] Nucleotide. A monomeric unit of DNA or RNA consisting of a sugarmoiety (pentose), a phosphate, and a nitrogenous heterocyclic base. Thebase is linked to the sugar moiety via the glycosidic carbon (1′ carbonof the pentose) and that combination of base and sugar is a nucleoside.The base characterizes the nucleotide. The four DNA bases are adenine(“A”), guanine (“G”), cytosine (“C”), and thymine (“T”). The four RNAbases are A, G, C, and uracil (“U”).

[0037] Operably Encodes or associated. Operably encodes or operablyassociated each refer to the functional linkage between a promoter andnucleic acid sequence, wherein the promoter initiates transcription ofRNA corresponding to the DNA sequence. A heterologous DNA sequence is“operatively associated” with the promoter in a cell when RNA polymerasewhich binds the promoter sequence transcribes the coding sequence intomRNA which is then in turn-translated into the protein encoded by thecoding sequence.

[0038] Phage or Bacteriophage. Bacterial virus many of which include DNAsequences encapsidated in a protein envelope or coat (“capsid”). In aunicellular organism a phage may be introduced by a process calledtransfection.

[0039] Plasmid. A non-chromosomal double-stranded DNA sequencecomprising an intact “replicon” such that the plasmid is replicated in ahost cell. When the plasmid is placed within a unicellular organism, thecharacteristics of that organism may be changed or transformed as aresult of the DNA of the plasmid. A cell transformed by a plasmid iscalled a “transformant.”

[0040] Polypeptide. A linear series of amino acids connected one to theother by peptide bonds between the alpha-amino and carboxy groups ofadjacent amino acids.

[0041] Promoter. A DNA sequence within a larger DNA sequence defining asite to which RNA polymerase may bind and initiate transcription. Apromoter may include optional distal enhancer or repressor elements. Thepromoter may be either homologous, i.e., occurring naturally to directthe expression of the desired nucleic acid, or heterologous, i.e.,occurring naturally to direct the expression of a nucleic acid derivedfrom a gene other than the desired nucleic acid. A promoter may beconstitutive or inducible.

[0042] Reading Frame. The grouping of codons during translation of mRNAinto amino acid sequences. During translation the proper reading framemust be maintained. For example, the DNA sequence may be translated viamRNA into three reading frames, each of which affords a different aminoacid sequence.

[0043] Recombinant DNA Molecule. A hybrid DNA sequence comprising atleast two DNA sequences, the first sequence not normally being foundtogether in nature with the second.

[0044] Ribosomal Binding Site. A nucleotide sequence of mRNA, coded forby a DNA sequence, to which ribosomes bind so that translation may beinitiated. A ribosomal binding site is required for efficienttranslation to occurs The DNA sequence coding for a ribosomal bindingsite is positioned on a larger DNA sequence downstream of a promoter andupstream from a translational start sequence.

[0045] Replicon. Any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo, i.e.,capable of replication under its own control.

[0046] Start Codon. Also called the initiation codon, is the first mRNAtriplet to be translated during protein or peptide synthesis andimmediately precedes the structural gene being translated. The startcodon is usually AUG, but may sometimes also be GUG.

[0047] Structural Gene. A DNA sequence which encodes through itstemplate or messenger RNA (mRNA) a sequence of amino acidscharacteristic of a specific polypeptide.

[0048] Transform. To change in a heritable manner the characteristics ofa host cell in response to DNA foreign to that cell. An exogenous DNAhas been introduced inside the cell wall or protoplast. Exogenous DNAmay or may not be integrated (covalently linked) to chromosomal DNAmaking up the genome of the cell. In prokaryotes and yeast, for example,the exogenous DNA may be maintained on an episomal element such as aplasmid. With respect to eucaryotic cells, a stably transformed cell isone in which the exogenous DNA has been integrated into a chromosome sothat it is inherited by daughter cells through chromosome replication.This stability is demonstrated by the ability of the eucaryotic cell toestablish cell lines or clones comprised of a population of daughtercells containing the exogenous DNA.

[0049] Transcription. The process of producing mRNA from a structuralgene.

[0050] Translation. The process of producing a polypeptide from mRNA.

DETAILED DESCRIPTION OF THE INVENTION

[0051] In the following description, the nomenclature used to define theproteins is that specified by Schroder and Lubke [“The Peptides,”Academic Press (1965)] wherein, in accordance with conventionalrepresentation, the N-terminal appears to the left and the C-terminal tothe right. Where the amino acid residue has isomeric forms, it is theL-form of the amino acid that is represented herein unless otherwiseexpressly indicated.

[0052] The invention can be used to insert any number or combination ofnative or foreign or heterologous DNA sequences into DNA vectors. TheseDNA sequences may be of any composition or function, they may encodeproteins, polypeptides, regulatory elements, promoters, markers, andother non-protein producing DNA, or RNA of interest from eukaryotic orprokaryotic sources or from viruses. Moreover, DNA sequences or theirencoded proteins, polypeptides, or RNA may have related functions, suchas different enzymes involved in a common metabolic pathway, or they maybe unrelated. Without being limited thereto, DNA sequences which may beincorporated into vectors for use herein may encode intracellularproteins, membrane proteins, and/or proteins secreted into the culturemedium. The DNA sequences may encode proteins of interest correspondingto all or part of native proteins found in nature. The encoded proteinsmay also include chimeric proteins, for example, fused polypeptides orthose from mutants displaying modified biological properties. Specificexamples of proteins of interest which may be encoded by the DNAsequences herein include: pharmaceuticals or veterinary agents such ascytokines, hormones, or anticoagulants, enzymes, enzyme inhibitors, andantigens or vaccines. In the preferred embodiment, the DNA sequenceswhich are used herein will encode a plurality of enzymes involved in acommon metabolic or biosynthetic pathway for converting a precursermolecule into a product of interest. Examples include but are notlimited to the enzymes of the carotenoid biosynthetic pathway asdescribed in the Examples hereinbelow.

[0053] A plurality of any of these DNA sequences may be sequentially anddirectionally cloned or inserted in any desired orientation into asingle vector. Furthermore, the sequences may be inserted in the sameorientation (relative to their direction of transcription) in thevector, or in different orientations such as when a promoter is includedwith each inserted sequence. However, without being limited thereto, theDNA sequences are preferably inserted into the vector in the sameorientation, with the direction of transcription (read from the 5′ endto the 3′ end of the coding strands) of each inserted sequence being thesame.

[0054] In accordance with this invention, the orientation of insertionof multiple DNA sequences may be controlled by use of restriction siteswhich have a degenerate recognition sequence and which generate cohesiveor overhanging ends upon cleavage by their corresponding restrictionendonuclease. As used herein, it is understood that the term“restriction sites which have a degenerate recognition sequence” refersto restriction sites having specific user defined sequences, and whichare cleaved by corresponding restriction endonucleases that recognizedegenerate internal sequences therein. These restriction sites are addedonto the ends of any two DNA sequences which are to be ligated, and thenucleotide sequences of the degenerate recognition sequences are chosenor tailored by the user such that the restriction sites will generatecohesive ends which are selectively complementary to each other. In thepreferred embodiment, when using DNA sequences which encode proteins ofinterest, these complementary restriction sites will be added onto theadjacent upstream and downstream ends of the two sequences. However, asdescribed above, it is also understood that the sequences may beinserted in the opposite orientation, with the downstream ends of eachsequence being adjacent, or with their upstream ends adjacent. Unlessotherwise specified, it is understood that in duplex DNA the terms“upstream” and “downstream” refer to the 5′ and 3′ directions from thecoding strand, respectively.

[0055] In general, whenever it is desired to ligate any second DNAsequence to a first DNA sequence, these restriction sites areselectively added onto the ends which are to be joined (preferably, butnot limited to their upstream and downstream ends, respectively). Bychoosing the nucleotide sequences of the degenerate recognitionsequences of these restriction sites such that the cohesive ends will becomplementary only to each other, the directionality of the ligation ofthe DNA sequences to one another will be ensured. As described herein,it is understood that the description of cohesive ends as“complementary” refers to the ends as having overhangs of the samelength and which are 100% complementary. Furthermore, as will bedescribed in greater detail hereinbelow, by using the same restrictionsite on the ends of the sequences, the functional restriction site maybe regenerated upon ligation into the vector, allowing its use for theinsertion of additional restriction sites. Conversely, by usingdifferent restriction sites on the ends of the sequences, a functionalrestriction site is not regenerated.

[0056] A variety of restriction sites which contain degeneraterecognition sequence and which generate cohesive ends upon cleavage asdescribed above are known and are suitable for use herein. Without beinglimited thereto, preferred restriction sites include: DraIII, SfiI,PfiMI, MwoI, BslI, BglI, and AlwNI. Each of these enzymes generate 3′cohesive ends with overhangs having a length of 3 nucleotides. Theirrecognition sequences and cleavage sites are as follows: EnzymeRecognition Sequence            ↓ DraIII 5′...CACNNNTGTG...3′3′...GTGNNNCAC...5′         ↑              ↓ SfiI5′...GGCCNNNNNGGCC...3′ 3′...CCGGNNNNNCCGG...5′           ↑            ↓ Pf1MI 5′...CCANNNNNTGG...3′ 3′...GGTNNNNNACC...5′         ↑             ↓ MwoI 5′...GCNNNNNNNGC...3′3′...CGNNNNNNNCG...5′          ↑             ↓ BslI5′...CCNNNNNNNGG...3′ 3′...GGNNNNNNNCC...5′          ↑             ↓BglI 5′...GCCNNNNNGGC...3′ 3′...CGGNNNNNCCG...5′          ↑            ↓AlwNI 5′...CAGNNNCTG...3′ 3′...GTCNNNGAC...5═         ↑

[0057] where the cleavage sites are indicated by the arrows. It isunderstood that the restriction sites are not limited to these examples,and other restriction sites containing degenerate recognition sequenceswhich generate cohesive ends of other lengths (such as overhangs of 4nucleotides) may also be used. The degenerate region of the recognitionsequence of any two of these sites (the same site or different sites)may be chosen such that they will generate selectively complementarycohesive ends upon cleavage. Corresponding restriction endonucleases arealso commercially available from a variety of sources. These restrictionenzymes typically cleave very infrequently in genomic DNA. However, theskilled practitioner will recognize that the specific restriction sitesselected for use should not appear within the DNA sequences of interest.

[0058] The first DNA sequence to be inserted into the vector may beinserted using conventional techniques. Insertion of the first DNAsequence does not itself require the use of the above-mentionedrestriction sites which contain a degenerate recognition sequence. Themanner in which the first DNA sequence is inserted is not critical, andwithout being limited thereto, may include ligation into vectorscontaining restriction sites generating blunt ends or cohesive ends uponcleavage, as described by Sambrook et al. (Molecular Cloning: Alaboratory Manual, Cold-Spring Harbor Laboratory Press, Plainview, N.Y.,1989), the contents of which are incorporated by reference herein. Forexample, in the preferred embodiment, the first DNA sequence of interestmay be provided with a single restriction site generating a cohesive endat one end thereof, and the vector provided with any first restrictionsite generating a complementary cohesive end and a second restrictionsite generating a blunt end. The restriction site on the DNA sequencegenerating the cohesive end should of course be located at theappropriate end thereof to ensure insertion in the desired orientation.Alternatively, the first DNA sequence may be inserted into the vector byproviding each of the vector and the ends of the DNA sequence with anyrestriction sites which generate complementary cohesive ends uponcleavage. When inserting the first DNA sequence in this manner, it ispreferred that the restriction sites on the ends of the DNA sequenceshould be different. The DNA sequence may then be inserted into thevector in the desired orientation by cleaving these sites with theircorresponding restriction enzymes, and ligating the resultantoverhanging and/or blunt ends.

[0059] In accordance with this invention, to allow the insertion of anynumber of additional sequences adjacent to (upstream and/or downstream)the first DNA sequence, each in a desired orientation, one of theabove-mentioned restriction sites which contain a degenerate recognitionsequence and which generate cohesive ends upon cleavage is addedadjacent to a selected end of the coding sequence of this first DNAsequence (i.e., at the end to which the additional sequences are to beadded). This restriction site is distinct from and should be differentfrom any restriction site(s) provided on the first DNA sequence to allowits insertion into the vector as described above. Moreover, it should benoted that if the first DNA sequence has any other restriction sites atthe same end (such as sites used for inserting the sequence into theplasmid), the restriction site containing the degenerate recognitionsequence must be positioned between those other sites and the DNA(coding) sequence.

[0060] To insert a second DNA sequence of interest adjacent to the firstDNA sequence, the second DNA sequence is also provided with arestriction site having a degenerate recognition sequence adjacent to afirst end thereof (i.e., the end which is to be adjacent to the selectedend of the first sequence described above) which, upon cleavage with itscorresponding restriction enzyme, generates a cohesive end. Thenucleotide sequences of the degenerate regions in each of theserestriction sites on the selected end of the first DNA sequence and thefirst end of the second DNA sequence are selected such that the cohesiveends generated adjacent to the selected end of the first DNA sequenceand the first end of the second DNA sequence upon cleavage, will becomplementary and anneal to each other. However, it is also noted thatwhile this restriction site on the first end of the second DNA sequenceshould also be selected from those restriction sites having andegenerate recognition sequence, it should not be the same as oridentical to the downstream restriction site on the first DNA sequence(e.g., if one is SfiI, the other could be any of DraIII, PfiMI, MwoI,BslI, BglI or AlwNI). By selecting different restriction sites thatgenerate complementary cohesive ends, during ligation of the first andsecond DNA sequences the ends will anneal but a functional restrictionsite will not be regenerated. This will prevent inadvertent cleavageduring the insertion of additional sequences.

[0061] If further DNA sequences are to be inserted adjacent to thesecond DNA sequence, the second DNA sequence should also include anadditional restriction site adjacent to its opposite or second end,which is essentially the same as or identical to the above-mentionedrestriction site on the selected end of the first DNA sequence. Asdefined herein, the restriction sites are “essentially the same” whenthey are the target sites for (cleaved by) the same restrictionendonuclease, and the nucleotide sequences of the portions of thedegenerate recognition sequences which form the cohesive ends uponcleavage are identical. It is envisioned that for those restrictionsites having degenerate recognition sequences which extend upstream ordownstream of the cleavage site (i.e., the degenerate recognitionsequences are longer than the cohesive or overhanging ends) thedegenerate nucleotides outside of the cleavage site may be different.Furthermore, the nucleotide sequences of the degenerate regions in theserestriction sites are selected such that the cohesive end on theselected end of the first DNA sequence is not complementary to thecohesive end on the second end of the second DNA sequence.

[0062] Digestion of each of the restriction sites on the ends of secondDNA sequence and the restriction site on the selected end of the firstDNA sequence (now contained within the recombinant vector) with theircorresponding restriction enzymes, and ligation of the resultantcohesive ends, will effectively insert the second DNA sequence into thevector adjacent to the first DNA sequence, oriented with the first endof the second sequence adjacent to the selected end of the firstsequence. Because the restriction sites at the opposite ends of thesecond DNA sequence are different, when they are cleaved they willgenerate asymmetric cohesive ends, and each cohesive end will becomplementary to only one of the ends on the digested vector.Directionality of the insertion in the desired orientation is thereforeensured.

[0063] Upon cleavage of the recombinant vector containing the first DNAsequence (at the restriction site at the selected end of the firstsequence) to insert the second DNA sequence, cohesive ends will becreated on both ends of the cut vector, one adjacent to the selected endof the first DNA sequence and the other at the opposite end of thevector. When the restriction sites on the second DNA sequence arecleaved, the restriction site adjacent to its first end will generate acohesive end which is only complementary to the cohesive end adjacent tothe first DNA sequence (at the selected end thereof), and therestriction site adjacent to its second end will generate a cohesive endwhich is only complementary to the cohesive end on the far end of thecut vector. When ligated, these cohesive ends will therefore not onlyanneal in the desired orientation, but the restriction site for the samerestriction enzyme will also be regenerated at the second end of thesecond DNA sequence. Recreation of this restriction site will allowinsertion of further DNA sequences adjacent to the second end of thesecond DNA sequence.

[0064] Any number of additional DNA sequences of interest may be addedfollowing the same protocol described for the second sequence. Forinstance, the third DNA sequence is also provided adjacent to a selectedfirst end with a restriction site having a degenerate recognitionsequence which generates a cohesive end upon cleavage with itscorresponding restriction enzyme. Again, as with the second DNAsequence, the restriction sites on the second end of the second DNAsequence and the first end of the third DNA sequence should bedifferent, but the nucleotide sequences of their degenerate regionswithin these restriction sites are selected such that the cohesive endsgenerated upon cleavage will be complementary and anneal to each other.As before, digestion of the third DNA sequence and the second DNAsequence (also now contained within the recombinant vector) with theircorresponding restriction enzymes and annealing the resultant cohesiveends, inserts the third DNA sequence into the vector adjacent to thesecond end of the second DNA sequence, again in the desired orientation.Furthermore, as described for the second DNA sequence, if more DNAsequences are to be inserted, the third DNA sequence should also includea restriction site adjacent to its opposite or second end which isidentical to the restriction site on the second end of the second DNAsequence. Still more DNA sequences may then be inserted in the samemanner.

[0065] Alternatively, if no further DNA sequences are to be inserted,the addition of a downstream restriction site on the last inserted DNAsequence, although preferred, is not critical and may even be optional.If omitted, the manner of annealing the downstream end of the last DNAsequence to the far end of the cut vector may be performed using avariety of techniques. For instance, a restriction site generating ablunt end may be provided in the vector upstream or downstream from thefrom the point of insertion of the above-mentioned DNA sequences.Cleavage with its corresponding restriction enzyme will therefore removethe overhang previously generated on the far end of the cut vector,allowing the second or free end of the last DNA sequence and vector toanneal. Conversely, any restriction site may be added to the second endof the last DNA sequence which, upon cleavage with its correspondingrestriction enzyme, generates a cohesive end which is complementary toand capable of annealing to the cohesive end on the far end of the cutvector. If no other DNA sequences are to be inserted, this restrictionsite need not be identical to the restriction site on the selected endof the first DNA sequence, but need only generate a complementarycohesive end.

[0066] In an alternative embodiment, it is also understood that additionof DNA sequences is not restricted to only one end of the first DNAsequence but that DNA sequences may be inserted adjacent to both ends ofthe first DNA sequence. Insertion of one or more further sequences atthe other end of the first DNA sequence is conducted using the sameprocess described above, with one of the above-mentioned restrictionsites having a degenerate recognition sequence being provided on thesecond end of the first DNA sequence (opposite from the selected end).This restriction site should of course be different from the restrictionsite at the selected end. The further DNA sequence to be inserted isthen provided with restriction sites in the same manner as theabove-described second DNA sequence, except that the restriction sitesat the ends of the further DNA sequence are selected relative to therestriction site at the second end of the first sequence.

[0067] The above-described restriction sites may be selectively addedonto the upstream and/or downstream end of any DNA sequence of interestusing Polymerase Chain Reaction (PCR) techniques or via incorporationusing oligonucleotide linkers with the desired sites. PCR techniques arewell known, and are described, for example, in Sambrook et al. (ibid),U.S. Pat. No. 4,683,195, and in Current Protocols in Molecular Biology,Ausubel et al. (eds.) Greene Publishing Assoc. And Wiley-Interscience,1991, volume 2, chapter 15. In the preferred embodiment, the 5′ and 3′PCR primers for the coding regions of the DNA sequences of interest aredesigned containing the selected restriction sites at their termini.Upon completion of amplification, the resultant DNA sequences willcontain the selected restriction sites at their upstream and downstreamends. Details of this process are described in the Examples.

[0068] The vector selected should possess appropriate restriction sitesfor insertion of the DNA sequences of interest. A large number ofvectors having polycloning sites are widely available and are suitablefor use herein. Within each specific vector, various restriction sitesmay be generally selected for insertion of the isolated DNA sequences.Alternatively, specific restriction sites of interest may be insertedinto a vector for subsequent cloning or expression of the DNA sequencesof interest, using known techniques such as described by Kleid et al.(U.S. Pat. No. 5,888,808).

[0069] The particular site chosen for insertion of the selected DNAsequences into the vector to form a recombinant vector is determined bya variety of factors recognized by those of skill in the art. None ofthese factors alone absolutely controls the choice of insertion site forparticular polypeptides. Rather, the site chosen reflects a balance ofthese factors, and not all sites may be equally effective for givenproteins.

[0070] The DNA sequences of the invention may be inserted into thedesired vector by known techniques. If the vector is to serve as anexpression vector, it should have or be provided with a promoter, andthe DNA sequences should be inserted in the vector downstream of thepromoter and operably associated or linked therewith. The promotershould be operable in the host cell into which it is to be inserted(that is, the promoter should be recognized by the RNA polymerase of thehost cell). While control sequences may be present with or ligated tothe DNA coding sequence(s) prior to insertion into the vector,alternatively, a vector may be selected which already possesses anoperable promoter. In addition, the vector may optionally have a regionwhich codes for a ribosome binding site positioned between the promoterand the site at which the DNA sequence is inserted so as to be operablyassociated with the DNA sequence of the invention once inserted (incorrect translational reading frame therewith). Moreover, the vectorshould preferably be selected to provide a region which codes for aribosomal binding site recognized by the ribosomes of the host cell intowhich the vector is to be inserted. The vector may also optionallycontain other regulatory sequences such as enhancer sequences,polyadenylation signals, transcription termination signals, orregulatory domains for effecting transcription and translation of theinserted coding sequences, or selectable markers as are known in theart, such as antibiotic resistance. The various DNA sequences ofinterest may be inserted with separate control elements, or under thecontrol of a single promoter. The former is generally-preferred whentransforming eukaryotic host cells.

[0071] In the preferred embodiment, the DNA-sequences of interest areinserted sequentially into the vector as described above and in theExamples. However, it is envisioned that the DNA sequences may be firstligated together in the desired order, and the resultant “cassette”inserted into the vector in a single step.

[0072] In an alternative embodiment, additional restriction sites may beprovided in the vector, one on each side flanking the region in whichthe DNA sequences are inserted. Upon completion of insertion of the DNAsequences, cleavage of these sites enables the ligated sequences to beextracted as a single “cassette” for insertion into a different vector.These additional restriction sites may be present in the vector prior toinsertion of the sequences, or they may be inserted with the first andlast inserted DNA sequences (e.g., one present upstream from the firstDNA sequence and the second present downstream from the last DNAsequence), or a combination thereof.

[0073] A variety of vector-host cell expression systems may beemployed-in practicing the present invention. Host cells may be eitherprocaryotic or eukaryotic, and, when the host cells are bacterial cells,they may be either gram-negative or gram-positive bacteria. Strains ofEscherichia coli are generally preferred for use in procaryotic systems.However, without being limited thereto, other useful hosts includespecies of Salmonella (including, for example, S. typhimurium, S.enteriditis, and S. dublin) species of Mycobacterium (such as M.smegmatis and M. bovis, species of Pseudomonas (including, for example,P. aeruginosa and P. putida), Bacillus subtilis, yeasts and other fungi(for example, Saccharomyces cerevisiae), plant cells such as plant cellsin culture (including, for example, both angiosperms and gymnosperms)and animal cells such as animal cells in culture.

[0074] Vectors used in practicing the present invention are selected tobe operable as cloning vectors or expression vectors in the selectedhost cell. While plasmid vectors are preferred, the vector may, forexample, be a virus, retrovirus, bacteriophage, cosmid, artificialchromosome (bacterial or yeast), or any nucleic acid sequence which isable to replicate in a host cell. Numerous vectors, including plasmids,are known to practitioners skilled in the art, and selection of anappropriate vector and host cell is a matter of choice. A number ofprocaryotic plasmid expression vectors are described in U.S. Pat. Nos.4,652,525, 4,440,859, 4,436,815, and 4,342,832, and a number ofeukaryotic plasmid expression vectors have also been described in U.S.Pat. Nos. 4,546,082, 4,510,245, 4,446,235, and 4,443,540. Further, thevectors may be non-fusion vectors (i.e., those producing polypeptides ofthe invention not fused to any heterologous polypeptide), oralternatively, fusion vectors (i.e., those producing the polypeptidefused to a vector encoded polypeptide). The fusion proteins would ofcourse vary with the particular vector chosen. Suitable non-fusionplasmid vectors for use with E. coli include but are not limited topTrc99 for use with E. coli JM 105, or pANK-12, pANH-1 or pPL2 for usewith E. coli MZ 1. Conversely, suitable fusion plasmid vectors includepGEX and pMC1871 for use with E. coli JM 105, pMAL with E. coli PR 722,pVB2 with E. coli LA5709, pTrcHis with E. coli INV F′, pC05 with E. coliN6405, and pRIT2T or pEZZ 18 with E. coli N4830-1. Other, non-E. coliexpression systems which may also be employed include pAc360 orpBluescript for use with SP2 or High 5 insect cells, pYesHis with theyeast S. cerevisiae INVSc1 or INVSc2, pLS405 with Salmonella dublinSL598, and pYUB12 with Mycobacterium smegmatis or M. bovis. Still othersuitable plasmid vector-host combinations that may be used in practicingthe instant invention are described, for example, in U.S. Pat. Nos.5,122,471 and 5,670,339 the contents of each of which are incorporatedby reference herein.

[0075] The proteins and polypeptides encoded by the inserted DNAsequences in this are expressed by growing host cells transformed by theexpression vectors described above under conditions whereby the proteinsor polypeptides are expressed. They may then be isolated from the hostcells if desired. Depending on the host cell used, transformation isdone using standard techniques. For example, the calcium treatmentemploying calcium chloride, described by Cohen (1972, Proc Natl Acad SciUSA, 69:2110), or the RbC1 method, described in Sambrook et al. (ibid)may be used for prokaryotes or other cells which contain substantialcell wall barriers. Infection with Agrobacterium tumefaciens such asdescribed by-Shaw (1983, Gene, 23:315) may be used for certain plantcells. For mammalian cells without such cell walls, the calciumphosphate precipitation method of Graham and Van der Eb (1978, Virology,52:546), or electroporation described in Sambrook et al. (ibid), may beused. Transformations into yeast may be conducted, for example,according to the method of Van Solingen, et al., (1977, J. Bacter.,130:946), and Hsiao et al. (1979, Proc Natl Acad Sci USA, 76:3829).

[0076] In general, after construction of a suitable expression vector,the vector is transformed into the appropriate host and successfultransformants may be selected by markers contained on the expressionvectors. Successfully transformed colonies are then cultured in order toproduce the proteins or polypeptides, or to produce cells having awell-defined number of copies of DNA elements of interest.

[0077] The following examples are intended only to further illustratethe invention and are not intended to limit the scope of the inventionwhich is defined by the claims.

EXAMPLE 1

[0078] To determine the utility of Tri5⁻ as a host strain for foreignisoprenoid production, we introduced three genes from the Erwiniauredovora carotenoid biosynthetic pathway into a strain of Fusariumsporotrichoides (NRRL 3299). In this strain the production oftrichothecenes, a family of toxic sesquiterpenoid epoxides, represents asignificant amount of total isoprenoid pathway carbon flow.Trichothecene production in F. sporotrichioides (NRRL 3299), underspecific growing conditions, can constitute as much as 10 to 20% ofculture fresh weight. This high level of isoprenoid pathway biosyntheticcapacity, if diverted from trichothecene production, could potentiallybe utilized in the production of commercially valuable isoprenoidcompounds.

[0079] The first committed step of trichothecene production, thecyclization of farnesyl diphosphate to form trichodiene, is catalyzed bythe sesquiterpene synthase trichodiene synthase (Tri5). The gene (andflanking sequences) coding for trichodiene synthase in F.sporotrichioides has been cloned and characterized (Hohn and Beremand,1989, Gene, 79:131-138). Using gene disruption of Tri5, a trichothecenedeficient mutant of NRRL 3299 has been generated (designated as Tri5⁻).This mutant strain of F. sporotrichioides afforded us with theopportunity to examine the feasibility of channeling isoprenoidprecursors formerly used for trichothecene biosynthesis into theproduction of heterologous isoprenoids.

[0080] Although trichothecene and carotenoid biosynthesis share thecommon precursor farnesyl diphosphate, the production of carotenoidsrequires the catalytic activity of enzymes not normally present in F.sporotrichioides, or which are expressed at very low levels. Tointroduce these genes via individual transformations would have beentechnically tedious, and the number of transformations limited by thesmall number of selectable markers available for use in this species. Inearlier work (Jones et al., 1996, “Metabolic engineering oftrichothecene-producing Fusarium”, Society for Industrial MicrobiologyAnnual Meeting Abstracts, 1996, P15), we discovered that NRRL 3299 (andTri5⁻) were resistant to the commonly used selective agents bialaphosand phleomycin, but were unable to grow on the alternate nitrogen sourceacetamide. This permitted us to utilize transformation methods using theAspergillus nidulans amdS gene as a selectable marker.

[0081] The process of this invention for the directional cloning ofmultiple genes was used to simultaneously introduce three genes requiredfor the synthesis of the carotenoid lycopene from farnesyl diphosphate.These genes, under the control of Tri5 promoter and terminator elements,produced functional enzymes when expressed in E. coli, and wereexpressed at high levels in certain Fusarium transformants.

Experimental Protocol

[0082] Cloning of Erwinia Crt Coding Regions

[0083] The following PCR primers were used to clone the crt genes fromErwinia genomic DNA: 5′TCCCCCGGGCAATATGACGGTCTGCGCAAAAAAACACG3′ (crtEsense), 5′CCGCTCGAGCATCCTTAACTGACGGCAGCG3′ (crtE antisense),5′TCCCCCGGGCAATATGGCAGTTGGCTCGAAAAGTT3′ (crbB sense),5′CCGCTCGAGGTTGTATATGGCGCACCGTATGC3′ (crtB antisense),5′TCCCCCGGGCAATATGAAACCAACTACGGTAATTGG3′ (crtI sense), and5′CCGCTCGAGACGGATTATTCAAATCAGATCCTCC3′ (crtI antisense).

[0084] antisense). The sense primers contained SmaI recognition sites,while the antisense primers contained XhoI recognition sites. PCRreactions were done according to manufacturer's protocol (Pfupolymerase, Stratagene, La Jolla, Calif.). Purification of PCR productswas done using Qiaquick spin columns (Qiagen, Santa Clarita, Calif.);following purification, DNA was digested with SmaI and XhoI (allrestriction enzymes obtained from New England Biolabs, Beverly, Mass.),and ligated into PmeI/XhoI digested pFSPME1. pFSPME1 was derived frompFS22-1 (Hohn and Beremand, ibid) by deletion of the Tris coding regionand introduction of PmeI and XhoI sites at the promoter and terminatortermini, respectively.

[0085] Assembly of Crt Cassette

[0086] Chimeric Tri5crtE was excised from PFSPME1 using the followingPCR primers: 5′GGAATTCGCGGCCGCTACAGATTCCCGCACAAAGGA3′ (CrtE5) and5′GGCCGAAAGGGCCAAACTCGGTGTAAAACAAGTTCCC3′ (Sfione).

[0087] Following purification of the PCR product, DNA was digested withEcoRI and ligated into EcoRI/SmaI digested BluescriptII KS⁻ (Stratagene,La Jolla, Calif.), producing pCRTEX1. Tri5crtB and Tri5crtI were excisedfrom pFSPME1 using the primers: 5′GCACTTTGTGAGTACAGATTCCCGCACAAAG3′(Draone) and Sfione. After digestion with DraII and SfiI, the two geneswere sequentially ligated into SfiI digested pCRTEX1, producing theplasmids pCRTEX2 and pCRTEX3. The crt cassette was excised from pCRTEX3by NotI digestion, and ligated into transformation vectors containingamdS (pA1L-E and pA4L-I) or hygB (pHL-J) selectable markers.

[0088] Protoplast Isolation and Transformation Protocols

[0089] Protoplast isolation and transformation of NRRL 3299 with thetransformation vector pHL-J followed the protocols described by Hohn, etal. (1993, Curr. Genet. 24:291-295). Protoplast isolation andtransformation of Tri5⁻ with the transformation vectors pA1L-E andpA4L-I followed the protocols described Royer et al. (1995,Bio/technology, 13:1479-1483) with the following modifications.Germinated spores were digested with a mixture of Novozyme 234 (5mg/mL), Driselase (25 mg/mL), and chitinase (0.05 mg/mL) in 0.7 m NaCl.Protoplasts were collected by centrifugation, washed twice with STC(1.4M sorbitol, 10 mM Tris-HCl pH 7.5, 50 mM CaCl₂), and diluted to aconcentration of 1×10⁸ protoplasts/mL in RSTC:SPTC:DMSO [8.0:2.0:0.1](RPTC=0.8M sorbitol, 50 mM Tris-HCl pH8, 50 mM CaCl₂; SPTC=40 % PEG4000, 0.8M sorbitol, 50 mM Tris-HCl pH8, 50 mM CaCl₂).

[0090] PCR Analysis of Transformants

[0091] PCR reactions were performed as per manufacturer'srecommendations (Taq polymerase, Promega, Madison, Wis.). To ascertainthe presence of the amdS selectable marker in E and I transformants, thefollowing primers were used: 5′GGGACTCGGTTCTGACAACC3′ (sense) and5′CCGAAATCGTGCTTGTATGG3′ (antisense). The anticipated product size withthese primers was approximately 700 bp. To determine the integrity ofthe introduced art gene cassette, the crtE sense and crtl antisenseprimers were utilized (product size 5.6 Kb). To determine the presenceof the individual Tri5crt genes in the transformants, Draone and therespective crt antisense primer were used in the PCR reaction.

[0092] DNA and RNA Analysis

[0093] DNA was isolated from cultures grown in YPG medium (0.3% yeastextract, 1% peptone, 2% glucose), following manufacturer's protocols(Genomic Tip-100, Qiagen, Santa Clarita, Calif.). RNA was isolated fromcultures grown in GYEP medium (5% glucose, 0.1% yeast extract, 0.1%peptone), following manufacturer's protocol (TRIZOL Reagent, GibcoBRLLife Technologies). Southern blotting was performed according to theprotocol of Hohn and Desjardins (Hohn and Desjardins, 1992, Mol.Plant-Microbe Interact., 5:249-256). RNA blotting was performed asdescribed by Proctor and Hohn (1993, J. Biol. Chem., 268:4543-4548).

[0094] Analysis of Lycopene Content

[0095] Liquid cultures were analyzed for lycopene by high-performanceliquid chromatography (HPLC). Mycelia filtered from 25-ml liquid shakecultures in GYEP medium were ground in liquid nitrogen in a mortar andpestle, and added to 50 ml Oakridge tubes containing 20 ml ofhexane:EtOH (2:1). Samples were incubated 30 minutes @ 37° C., andlayers separated by centrifugation. The organic phase was-removed, driedunder nitrogen gas stream, and reconstituted in 500 μL methyl tert-butylether (MTBE). Lycopene was detected by HPLC using a YMC Carotenoid C30reverse-phase column. Lycopene was eluted using a mobile phase ofmethanol/MTBE, with a gradient of 30-75% MTBE in 40 minutes. Flow ratewas 1.0 ml/minute. Detection of lycopene was done at 470 nm using aSpectra flow 783 Programmable Absorbance Detector (Kratos Division, ABIAnalytical, Ramsey, N.J.).

Results and Discussion

[0096] Construction of Chimeric Carotenoid Biosynthetic Genes

[0097] The carotenoid biosynthetic genes introduced into F.sporotrichioides were crtE (geranylgeranyl pyrophosphate synthase), crtB(phytoene synthase), and crtI (phytoene desaturase). The enzymes encodedby these genes catalyze the conversion of two molecules of farnesyldiphosphate into a molecule of the carotenoid lycopene. The codingregions for these enzymes were isolated from the Erwinia uredovora crtcluster (Misawa et al., J. of Bact., 172:6704-6712), utilizing thepolymerase chain reaction or PCR (primers and PCR conditions detailed inthe Experimental Protocol). The PCR primers used included therecognition sites for the restriction enzymes SmaI (on the senseprimers) and XhoI (on the antisense primers); following purification,the PCR products were digested with SmaI and XhoI and directionallycloned into PmeI/XohI digested pFSPME1 (FIG. 2). This plasmid containsboth the 5′ and 3′ flanking regions of Tri5 from F. sporotrichioides.This procedure was done for all three Erwinia crt coding regions,producing chimeric versions of crtE, crtB, and crtI (Tri5crtE, Tri5crtB,and Tri5crtI). Each chimeric gene possessed approximately 730 bp of theTri5 promoter, and 360 bp of 3′ sequence (including the transcriptionstop signal).

[0098] Design of Compatible DraIII/SfiI Overhangs

[0099]FIG. 1 illustrates the DraIII and SfiI sites that were designedfor the primers used in our cloning strategy. PCR primers containingsequences complementary to the desired target are designed containingDraIII and SfiI sites at the 5′ and 3′ ends, respectively (shown inTable 1). The 3′ AAA extension generated by DraIII extension can only beligated to the 3′ (TTT) extension of a digested SfiI site on theplasmid, thus ensuring the directionality of the insertion event. Otherextensions could also be designed to give the desired result (i.e., CAC,TCT, CAT), but this was the nucleotide sequence used in our studies. Theligation-of the DraIII cohesive end with a SfiI cohesive end produces asequence uncleavable by either enzyme; however, the ligation of the twoSfiI cohesive ends regenerates a sequence recognizable by SfiI.

[0100] Creation of the Gene Cassette

[0101] There are two options as to how the gene cassette is initiated.One method would be to simply introduce the first insert (with DraIIIand SfiI sites at the 5′ and 3′ termini, respectively) into a vectordigested with a restriction enzyme that produces blunt ends (e.g.,EcoRV). Further inserts could then be added by digesting the insertswith DraIII and SfiI, followed by ligation into SfiI-digested vector.Depending on the availability of restriction sites flanking the growingcassette, it may be problematic when attempting to move the cassettefrom one vector to another. To avoid this problem, we utilized thestrategy outlined in FIG. 3. The first insert was amplified usingprimers which contained EcoRI and NotI sites on the primer for the 5′end of the insert (Eco/Not), while a SfiI site was present on the primerfor the 3′ end of the insert (Sfione). This PCR product was digestedwith EcoRI , and cloned into EcoRI/SmaI digested BluescriptII KS⁻. Thisallowed us to take advantage of the NotI site present in the MCS of theplasmid; by adding an additional NotI site via PCR, we were able toflank the cassette with NotI sites, which could be utilized to excisethe cassette and move it into alternate vectors (NotI is not present inany of our insert sequences).

[0102] Once the first insert is in place in the vector, additionalinserts can be added by digesting the inserts with DraIII and SfiI, andcloning the fragments into the SfiI-digested vector. Upon ligation ofinsert and vector, a SfiI site is preserved at the 3′ end of thecassette, and additional inserts can be added as shown. To date, we havesuccessfully cloned 4 genes (comprising approximately 9 Kb) in tandem ina single vector. Although our inserts share a great deal of repeatedsequence (due to possessing the same promoter and terminator sequences),we have not observed any problems with recombination within similarsequences while the plasmids are being maintained in E. coli.Furthermore, we have excised the gene cassette (using NotI) andintroduced it into a number of transformation vectors used in ourlaboratory; this is a tremendous improvement over having to clone thesequences into each vector independently. Using this cloning strategyalso reduces the number of transformations necessary to introduce thedesired genes into a target organism (Fusarium sporotrichioides in ourcase); this is especially important when only a limited number ofselectable markers are available for transformations.

[0103] After the insertion of Tri5crtE, Tri5crtB, and Tri5crtI, the crtgene cassette was excised by NotI digestion, and cloned intotransformation vectors containing either amdS (for Tri5⁻) or hygB (forNRRL 3299) selectable markers. The resultant plasmids, designatedpA1L-E, pA4L-1, and pHL-J, are depicted in FIG. 4.

[0104] Analysis of Fusarium Transformants

[0105] Using transformation protocols detailed in the ExperimentalProtocol, the transformation vectors carrying the crt cassette wereintroduced into competent F. sporotrichioides protoplasts, resulting in6 transformants from NRRL 3299 protoplasts (J transformants), and 11transformants from Tri5⁻ protoplasts (E and I transformants).Preliminary analysis of the primary transformants indicated a high levelof untransformed nuclei were present in the amdS transformants (E andI). Single spore purification of the primary transformants was necessaryto examine whether the introduced genes were being properly expressed.This purification was accomplished for all but two of the E transformantlines. PCR analysis demonstrated that the art gene cassette wasintegrated as an intact unit in 6 of the transformants, with theremaining transformants (with the exception of J6) missing either one ortwo of the introduced genes, presumably due to various recombinationevents which occurred between the trichodiene synthase elements of thechimeric genes and the endogenous trichodiene synthase sequences inFusarium. Interestingly, transformant J6 contained all three introducedgenes, but not as an intact unit. Southern analysis confirmed theresults of the PCR analysis.

[0106] RNA analysis of single spore purified transformants demonstratedthe presence of Tri5crtE, Tri5 crtB, and Tri5 crtI transcripts(corresponding to geranylgeranyl-pyrophosphate synthase, phytoenesynthase, and lycopene cyclase, respectively). Expression of transgeneswas similar to that of Tri4 expression. Tri4, a cytochrome P450monooxygenase involved in trichothecene biosynthesis (Hohn et al., 1995,Mol. Gen. Genet., 248:95-102), is expressed at levels similar to Tri5under the culture conditions used. This result demonstrated that theTri5 promoter used in creating Tri5crtE, Tri5crtB, and Tri5crtI was ofsufficient size to obtain optimal transgene expression. Previouslystudies have demonstrated that Tri5 (and Tri4) RNA levels are inducedover 50-fold after 24 h of culture of NRRL 3299 in GYEP medium. Thepattern of transgene expression correlated with earlier PCR data (Joneset al., 1996, Metabolic Engineering of Trichothecene-producing Fusarium,Society for Industrial Microbiology Annual Meeting Abstracts, P15); thenumber of transgene transcripts equaled the number of carotenoidbiosynthetic genes that were detected within each individualtransformant.

[0107] Analysis of Carotenoid Expression in E. coli and FusariumTransformants

[0108] Lycopene production was observed in crt gene cassette-carrying E.coli, and in Fusarium transformants. Lycopene production was firstobserved after 3 days of culture, with maximum production observed in 5to 6 day old cultures (˜0.5 mg/g culture dry weight).

[0109] Four of the preferred lycopene producing strains of transformedFusarium sporotrichioides, designated E22e, I8d, I91a, and J62, wereretained. All four strains have been deposited under the Budapest Treatyin the United States Department of Agriculture Agricultural ResearchService culture collection in Peoria, Ill., on Jul. 1, 1999, and havebeen assigned deposit accession numbers NRRL ______.

[0110] In summary, we have developed a strategy using complementaryDraIII/SfiI restriction sites to directionally clone multiple genes intoFusarium sporotrichioides, with each gene under the control of an active(Tri5) Fusarium-specific promoter. We are currently expanding the sizeof our cassette, thereby increasing the number of carotenoid productswhich can be produced. In our current study, approximately 40% (6/17)transformants contained an intact crt gene cassette. It is reasonable toassume that as the number of genes in the cassette increases, thelikelihood of trichodiene synthase elements recombination withendogenous sequences also increases, leading to loss of one or more ofthe introduced genes. We have been able to demonstrate that the chimericgenes are capable of producing functional enzymes, and high levels ofchimeric gene expression were observed in some of the transformants. Theresults of this study demonstrate the feasibility of geneticallyengineering Fusarium sporotrichioides in order to utilize the species asa host for high level synthesis of commercially valuable isoprenoidproducts.

EXAMPLE 2

[0111] To increase the biosynthetic capabilities of Tri5-beyond lycopeneproduction, a new cassette was constructed by the addition of crtY(downstream of the crtI gene in the cassette of Example 1), an Erwiniauredovora gene encoding for lycopene cyclase. This new, four genecassette should permit the production of β-carotene in transformedFusarium. CrtY was modified and inserted into the cassette as follows;the gene coding sequence was amplified from E. uredovora genomic DNAusing Pfu polymerase and the following primers: CRTY5 (5′CCCGGGCAATATGCAACCGCATTATGATCTGATTC 3′) and YREV6 (5′CGCTCGAGCCGTAGTTGGTTTCATGTAGTCGC 3′). After digestion with SmaI andXhoI, the fragment was ligated into pFSPME1. The chimeric crtY gene wasthen amplified using Pfu polymerase and Draone and Sfione primers, andafter DraIII/SfiI digestion, the crtY gene was ligated into the existingthree gene cassette.

[0112] The new four gene cassette was liberated from Bluescript usingNotI digestion, and the cassette was ligated into a second plasmid,pAMDS4, to form pBCX-14. The cassette was also ligated into a newexpression vector. This new vector, constructed from pAMDS4, containsTri10 (upstream of the β-carotene cassette), a gene present in theFusarium trichothecene gene cluster, which appears to have a role inregulating and enhancing expression of other Tri genes. Using PCRprimers (#247: 5′GGTCAACATGATGTCAGG 3′; #620: 5′CGCCAAGTACGTGGACCGGCTGCACATGTCAAGG 3′), the Tri10 gene was amplifiedfrom pTRI9D4-7 and inserted into pAMDS4. Upon addition of the four genecassette, the resultant plasmid was named pTRIBCX-44.

[0113] pBCX-14 and pTRIBCX-44 were transformed into Fusariumsporotrichioides (Tri5−) protoplasts as previously described. Fourindependent transformants containing pBCX-14 (FS1-4) and 3 independenttransformants containing pTRIBCX-44 (ST1-3) were single spore purifiedand analyzed further. The four gene cassette was found to be integratedas an intact unit in all seven transformants. β-carotene production wasobserved in FS and ST transformant strains. The greatest carotenoidproduction was observed in ST transformants, with yields reaching 3 to 4mg β-carotene per gram of fungus (dry weight).

[0114] Three of the preferred β-carotene producing strains oftransformed Fusarium sporotrichioides, designated ST1, ST2, and ST3,were retained. All three strains have been deposited under the BudapestTreaty in the United States Department of Agriculture AgriculturalResearch Service culture collection in Peoria, Ill., on Jul. 15, 1999,and have been assigned deposit accession numbers NRRL ______.

[0115] It is understood that the foregoing detailed description is givenmerely by way of illustration and that modifications and deviations maybe made therein without departing from the spirit and scope of theinvention. TABLE 1 Oligonucleotides added to insert sequence specificPCR primers for the purpose of introducing restriction endonucleasesites Primer Name Added sequence Notes Eco/Not 5′GGAATTCGCGGCCGC3′Primer for 5′end of first insert Draone 5′GCACTTTGTGAG3′ Primer for5′end of remaining inserts Sfione 5′GGCCGAAAGGGCC3′ Primer for 3′ends ofinserts

[0116]

1 26 1 38 DNA Erwinia uredovora 1 tcccccgggc aatatgacgg tctgcgcaaaaaaacacg 38 2 30 DNA Erwinia uredovora 2 ccgctcgagc atccttaactgacggcagcg 30 3 35 DNA Erwinia uredovora 3 tcccccgggc aatatggcagttggctcgaa aagtt 35 4 32 DNA Erwinia uredovora 4 ccgctcgagg ttgtatatggcgcaccgtat gc 32 5 36 DNA Erwinia uredovora 5 tcccccgggc aatatgaaaccaactacggt aattgg 36 6 34 DNA Erwinia uredovora 6 ccgctcgaga cggattattcaaatcagatc ctcc 34 7 37 DNA Fusarium sporotrichioides 7 ggaattcgcggccgctacag attcccgcac aaaggaa 37 8 37 DNA Fusarium sporotrichioides 8ggccgaaagg gccaaactcg gtgtaaaaca agttccc 37 9 31 DNA Fusariumsporotrichioides 9 gcactttgtg agtacagatt cccgcacaaa g 31 10 20 DNAAspergillus sp. 10 gggactcggt tctgacaacc 20 11 20 DNA Aspergillus sp. 11ccgaaatcgt gcttgtatgg 20 12 35 DNA Erwinia uredovora 12 cccgggcaatatgcaaccgc attatgatct gattc 35 13 32 DNA Erwinia uredovora 13 cgctcgagccgtagttggtt tcatgtagtc gc 32 14 18 DNA Fusarium sporotrichioides 14ggtcaacatg atgtcagg 18 15 34 DNA Fusarium sporotrichioides 15 cgccaagtacgtggaccggc tgcacatgtc aagg 34 16 13 DNA Fusarium sporotrichioides 16ggccctttcg gcc 13 17 13 DNA Streptomyces fimbriatus variation (5)..(9) nmay be A, T, G, or C 17 ggccnnnnng gcc 13 18 13 DNA Streptomycesfimbriatus variation (5)..(9) n may be A, T, G, or C 18 ggccnnnnng gcc13 19 11 DNA Pseudomonas fluorescens variation (4)..(8) n may be A, T, Gor C 19 ccannnnntg g 11 20 11 DNA Pseudomonas fluorescens variation(4)..(8) n may be A, T, G or C 20 ccannnnntg g 11 21 11 DNAMethanobacterium wolfei variation (3)..(9) n may be A, T, G or C 21gcnnnnnnng c 11 22 11 DNA Methanobacterium wolfei variation (3)..(9) nmay be A, T, G or C 22 gcnnnnnnng c 11 23 11 DNA Bacillus sp. variation(3)..(9) n may be A, T, G or C 23 ccnnnnnnng g 11 24 11 DNA Bacillus sp.variation (3)..(9) may be A, T, G or C 24 ccnnnnnnng g 11 25 11 DNABacillus globigii variation (4)..(8) n may be A, T, G or C 25 gccnnnnnggc 11 26 11 DNA Bacillus globigii variation (4)..(8) may be A, T, G or C26 gccnnnnngg c 11

We claim:
 1. A method for directionally inserting DNA sequences into avector comprising: a) providing a recipient DNA vector having a firstrestriction site therein, wherein said first restriction site contains adegenerate recognition sequence and which generates cohesive ends whendigested with its corresponding restriction endonuclease; b) digestingsaid vector with said restriction endonuclease corresponding to saidfirst restriction site, said first restriction site generating first andsecond cohesive ends on the digested vector; c) providing a first insertDNA segment comprising a first target DNA sequence having second andthird restriction sites adjacent to its first and second ends,respectively, wherein said second restriction site is different fromsaid first restriction site of said vector, contains a degeneraterecognition sequence and generates a cohesive end when digested with itscorresponding restriction endonuclease, wherein the nucleotide sequenceof said degenerate recognition sequence in said second restriction sitehas been selected such that the cohesive end generated from digestion ofsaid second restriction site is complementary to said first cohesive endon the digested vector of (b); and wherein said third restriction siteis essentially the same as said first restriction site of said vector,contains a degenerate recognition sequence and generates a cohesive endwhen digested with its corresponding restriction endonuclease, whereinthe nucleotide sequence of said degenerate recognition sequence in saidthird restriction site has been selected such that the cohesive endgenerated from digestion of said third restriction site is complementaryto said second cohesive end on the digested vector of (b), and whenannealed thereto regenerates a restriction site on the second end ofsaid target DNA sequence which is essentially the same as said thirdrestriction site; d) digesting said first insert DNA segment with saidrestriction endonucleases corresponding to said second restriction siteand said third restriction site; e) ligating the digested vector of (b)and the digested first insert DNA segment of (d) to produce arecombinant vector comprising said first target DNA sequence, with arestriction site on the second end of said first target DNA sequencebeing regenerated within said recombinant vector which is essentiallythe same as said third restriction site.
 2. The method of claim 1wherein said recipient DNA vector of (a) comprises a vector havinginserted therein an initial DNA sequence having said first restrictionsite adjacent to a selected end thereof, and said recombinant vector of(e) comprises said initial DNA sequence and said first target DNAsequence, with said first end of said first target DNA sequence beingadjacent to said selected end of said initial DNA sequence.
 3. Themethod of claim 1 further comprising f) digesting said recombinantvector with said restriction enzyme corresponding to said regeneratedrestriction site of (e), generating first and second cohesive ends onsaid recombinant vector, said first cohesive end being on the end of thecleaved vector adjacent to said second end of said first target DNAsequence, and said second cohesive end being on the opposite end of thecleaved vector; g) providing a second insert DNA segment comprising asecond target DNA sequence having fourth and fifth restriction sitesadjacent to its first and second ends, respectively, wherein said fourthrestriction site is different from said regenerated restriction site of(e), and also contains a degenerate recognition sequence and whichgenerates a cohesive end when digested with its correspondingrestriction endonuclease, wherein the nucleotide sequence of saiddegenerate recognition sequence in said fourth restriction site has beenselected such that said cohesive end generated from digestion of saidfourth restriction site is complementary to said first cohesive end onthe digested recombinant vector of (f); and wherein said fifthrestriction site is essentially the same as said first restriction siteof said vector, and also contains a degenerate recognition sequence andwhich generates a cohesive end when digested with its correspondingrestriction endonuclease, wherein the nucleotide sequence of saiddegenerate recognition sequence in said fifth restriction site has beenselected such that said cohesive end generated from digestion of saidfifth restriction site is complementary to said second cohesive end onthe digested recombinant vector of (f), and when annealed theretoregenerates a restriction site adjacent to the second end of said thirdDNA sequence which is essentially the same as said fifth restrictionsite; h) digesting said second insert DNA segment with said restrictionendonucleases corresponding to said fourth restriction site and saidfifth restriction site; i) ligating the digested recombinant vector of(f) and the digested second insert DNA segment of (h) to produce asecond recombinant vector comprising said first target DNA sequence andsaid second target DNA sequence, with said first end of said secondtarget DNA sequence being adjacent to said second end of said firsttarget DNA sequence, and a restriction site adjacent to the second endof said second target DNA sequence being regenerated within said secondrecombinant vector which is essentially the same as said fifthrestriction site.
 4. The method of claim 1 wherein said first, second,third, fourth, and fifth restriction enzymes are selected from the groupconsisting of SfiI, MwoI, PflI, DraIII, AlwNI, BglI, and BslI.
 5. Themethod of claim 1 wherein said first target DNA sequence is downstreamof and operably associated with a promoter.
 6. The method of claim 5,wherein said first insert DNA segment further comprises a promoterupstream of and operably associated with said first target DNA sequence,and said second insert DNA segment further comprises a promoter upstreamof and operably associated with said second target DNA sequence.
 7. Themethod of claim 5 wherein vector of (a) is an expression vectorcontaining said promoter, said promoter being upstream from the point ofinsertion of said first insert DNA segment.
 8. The method of claim 2wherein said recombinant vector further comprises a first additionalrestriction site adjacent to the end of said initial DNA sequenceopposite from said selected end, and a second additional restrictionsite adjacent to said second end of said first target DNA sequence andexternal to said regenerated restriction site of (e).
 9. The process ofclaim 8 further comprising digesting said recombinant vector of (e) withthe restriction endonuclease corresponding to said first and secondadditional restriction sites to excise an expression cassette comprisingsaid initial DNA sequence linked to said first target DNA sequence. 10.The method of claim 3 wherein said second recombinant vector of (i)further comprises a first additional restriction site adjacent to saidfirst end of said first target DNA sequence, and a second additionalrestriction site adjacent to said second end of said second target DNAsequence and external to said regenerated restriction site of (e). 11.The process of claim 10 further comprising digesting said secondrecombinant vector of (i) with the restriction endonucleasecorresponding to said first and second additional restriction sites toexcise an expression cassette comprising said first target DNA sequencelinked to said second target DNA sequence.
 12. The method of claim 3wherein said first and second target DNA sequences encode first andsecond proteins or polypeptides.
 13. The method of claim 12 wherein saidfirst and second target DNA sequences encode first and second enzymes.14. The method of claim 12 wherein said first and second target DNAsequences within said second recombinant vector of (i) are downstream ofand operably associated with a promoter.
 15. The method of claim 1wherein said vector is a plasmid.
 16. A method for preparing multiplerecombinant proteins or polypeptides of interest comprising: a)transforming host cells with the second recombinant vector of claim 14;b) culturing said cells under conditions effective for the expression ofsaid first and second proteins or polypeptides.
 17. The method of claim16 wherein said host cells are selected from the group consisting ofeukaryotic cells and prokaryotic cells.
 18. A method for the sequentialand directional cloning of multiple DNA sequences into a single vectorcomprising: a) providing a vector having one or more restriction siteseffective for insertion of DNA therein; b) providing a first DNA segmentcomprising a first DNA sequence of interest having a first restrictionsite adjacent to a selected end, wherein said first restriction sitecontains a degenerate recognition sequence and which generates acohesive end when digested with its corresponding restrictionendonuclease; c) inserting said first DNA segment into said vector toproduce a first recombinant vector comprising said first DNA sequencehaving said first restriction site adjacent to its selected end; d)digesting said first recombinant vector with said restrictionendonuclease corresponding to said first restriction site, said firstrestriction site generating first and second cohesive ends on saidrecombinant vector, said first cohesive end being on the end of thecleaved vector adjacent to said selected end of said first DNA sequence,and said second cohesive end being on the opposite end of the cleavedvector; e) providing a second DNA segment comprising a second DNAsequence of interest having second and third restriction sites adjacentto its first and second ends, respectively, wherein said secondrestriction site is different from said first restriction site of saidfirst DNA segment, and also contains a degenerate recognition sequenceand which generates a cohesive end when digested with its correspondingrestriction endonuclease, wherein the nucleotide sequence of saiddegenerate recognition sequence in said second restriction site has beenselected such that said cohesive end generated from digestion of saidsecond restriction site is complementary to said first cohesive end onthe digested vector of (d); and wherein said third restriction site isessentially the same as said first restriction site of said first DNAsegment, and also contains a degenerate recognition sequence and whichgenerates a cohesive end when digested with its correspondingrestriction endonuclease, wherein the nucleotide sequence of saiddegenerate recognition sequence in said third restriction site has beenselected such that said cohesive end generated from digestion of saidthird restriction site is complementary to said second cohesive end onthe digested vector of (d), and when annealed thereto regenerates arestriction site adjacent to the second end of said second DNA sequencewhich is essentially the same as said third restriction site; f)digesting said second DNA segment with said restriction endonucleasescorresponding to said second restriction site and said third restrictionsite; g) ligating the digested recombinant vector of (d) and thedigested second DNA segment of (f) to produce a second recombinantvector comprising said first DNA sequence and said second DNA sequence,with said first end of said second DNA sequence being adjacent to saidselected end of said first DNA sequence and a restriction site adjacentto the second end of said second DNA sequence being regenerated withinsaid second recombinant vector which is essentially the same as saidthird restriction site.
 19. The method of claim 18 further comprising:h) digesting said second recombinant vector with said restriction enzymecorresponding to said regenerated restriction site of (g), generatingfirst and second cohesive ends on said second recombinant vector, saidfirst cohesive end being on the end of the cleaved vector adjacent tosaid second end of said second DNA sequence, and said second cohesiveend being on the opposite end of the cleaved vector; i) providing athird DNA segment comprising a third DNA sequence of interest havingfourth and fifth restriction sites adjacent to its first and secondends, respectively, wherein said fourth restriction site is differentfrom said regenerated restriction site of (g), and also contains adegenerate recognition sequence and which generates a cohesive end whendigested with its corresponding restriction endonuclease, wherein thenucleotide sequence of said degenerate recognition sequence in saidfourth restriction site has been selected such that said cohesive endgenerated from digestion of said fourth restriction site iscomplementary to said first cohesive end on the digested secondrecombinant vector of (h); and wherein said fifth restriction site isessentially the same as said first restriction site of said first DNAsegment, and also contains a degenerate recognition sequence and whichgenerates a cohesive end when digested with its correspondingrestriction endonuclease, wherein the nucleotide sequence of saiddegenerate recognition sequence in said fifth restriction site has beenselected such that said cohesive end generated from digestion of saidfifth restriction site is complementary to said second cohesive end onthe digested second recombinant vector of (h), and when annealed theretoregenerates a restriction site adjacent to the second end of said thirdDNA sequence which is essentially the same as said fifth restrictionsite; j) digesting said third DNA segment with said restrictionendonucleases corresponding to said fourth restriction site and saidfifth restriction site; k) ligating the digested second recombinantvector of (h) and the digested third DNA segment of (j) to produce athird recombinant vector comprising said first DNA sequence, said secondDNA sequence, and said third DNA sequence, with said first end of saidsecond DNA sequence being adjacent to said selected end of said firstDNA sequence, said first end of said third DNA sequence being adjacentto said second end of said second DNA sequence, and a restriction siteadjacent to the second end of said third DNA sequence being regeneratedwithin said third recombinant vector which is essentially the same assaid fifth restriction site.
 20. The method of claim 18 wherein saidfirst, second, third, fourth, and fifth restriction enzymes are selectedfrom the group consisting of SfiI, MwoI, PflI, DraIII, AlwNI, BglI, andBslI.
 21. The method of claim 18 wherein said first and second DNAsequences are downstream of and operably associated with a promoter. 22.The method of claim 21, wherein said first DNA segment further comprisesa promoter upstream of and operably associated with said first DNAsequence, and said second DNA segment further comprises a promoterupstream of and operably associated with said second DNA sequence. 23.The method of claim 21 wherein vector of (a) is an expression vectorcontaining said promoter, said promoter being upstream from the point ofinsertion of said first DNA segment.
 24. The method of claim 18 whereinsaid second recombinant vector further comprises a first additionalrestriction site adjacent to the end of said first DNA sequence oppositefrom said selected end, and a second additional restriction siteadjacent to said second end of said second DNA sequence and external tosaid regenerated restriction site of (g).
 25. The process of claim 24further comprising digesting said second recombinant vector of (g) withthe restriction endonuclease corresponding to said first and secondadditional restriction sites to excise an expression cassette comprisingsaid first DNA sequence linked to said second DNA sequence.
 26. Themethod of claim 18 wherein said first and second DNA sequences encodefirst and second proteins or polypeptides.
 27. The method of claim 26wherein said first and second DNA sequences encode first and secondenzymes.
 28. The method of claim 26 wherein said first and second DNAsequences within said second recombinant vector of (g) are downstream ofand operably associated with a promoter.
 29. The method of claim 18wherein said vector is a plasmid.
 30. A method for preparing multiplerecombinant proteins or polypeptides of interest comprising: a)transforming host cells with the second recombinant vector of claim 28;b) culturing said cells under conditions effective for the expression ofsaid first and second proteins or polypeptides.
 31. The method of claim30 wherein said host cells are selected from the group consisting ofeukaryotic cells and prokaryotic cells.
 32. A transformed strain ofFusarium sporotrichioides effective for production of lycopene selectedfrom the group consisting of E22e, I8d, I91a, and J62.
 33. A transformedstrain of Fusarium sporotrichioides effective for production ofβ-carotene selected from the group consisting of ST1, ST2, and ST3.